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At least 19 recordsLinked to original sources
PubMed1990

Interleukin 1 receptors on rabbit articular chondrocytes: relationship between biological activity and receptor binding kinetics.

This study gives an account of the biologic and kinetic binding properties of interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta), and Glu-4 (an NH2-terminal mutant of IL 1 beta) to interleukin 1 (IL 1) receptors in rabbit articular chondrocytes. All three IL 1's demonstrated full agonist properties in their ability to stimulate prostaglandin E2 (PGE2) synthesis. IL 1 alpha was 23-fold more biologically active than IL 1 beta, which was around 110-fold more active than Glu-4 based on the concentration of IL 1 required for half-maximal stimulation of PGE2. The binding of all three ligands was concentration-dependent and saturable at 4 degrees C. Scatchard analysis of receptor binding data showed that the dissociation constant (KD) of IL 1 alpha was 46 +/- 12 pM, and the receptor density was 3120 sites/cell. The association of IL 1 alpha at 4 degrees C did not attain equilibrium until after 10 h at 100 pM of 125I-labeled IL 1 alpha. The dissociation of bound IL 1 alpha was very slow, t1/2 of 21 h, although only one class of high-affinity receptors was detected. The KD of IL 1 beta binding was 72 +/- 3 pM with a receptor density of 800 +/- 40 sites/cell. Dissociation of bound 125I-labeled IL 1 beta at 4 degrees C appeared to indicate the presence of two receptor subsets, a fast and a slower component with a t1/2 of 2 min and 5 h, respectively. The receptor binding affinity of Glu-4 was 324 +/- 3 pM, in line with its reduced biologic activity. Both IL 1 alpha and IL 1 beta are rapidly internalized in chondrocytes in a time- and temperature-dependent manner.

Animals↗
PubMed1992

The interleukin-1 receptor in normal and osteoarthritic human articular chondrocytes. Identification as the type I receptor and analysis of binding kinetics and biologic function.

OBJECTIVE: To identify and investigate the kinetic binding properties of interleukin-1 receptors (IL-1R), and examine the abilities of the 2 IL-1 isoforms to stimulate metalloprotease synthesis, in normal and osteoarthritic (OA) chondrocytes. METHODS: Receptor affinity and density were determined using radioligand binding experiments and flow cytometry. Immunocytochemical analysis and affinity cross-linking studies were performed for characterization of IL-1R. RESULTS: While no difference in receptor affinity between normal and OA chondrocytes was noted in binding studies (Kd approximately 30 pM), a 2-fold increase in receptor density was found in OA chondrocytes as compared with normal chondrocytes (mean 4,069 sites/cell versus 2,315 sites/cell). Flow cytometry experiments also showed a significant increase in receptor density in OA cells, as well as an enhancement in the percentage of positive cells in diseased cartilage compared with normal. Binding data for both IL-1 isoforms revealed a single class of binding sites and receptor specificity. Factors such as IL-2, interferon-gamma, tumor necrosis factor alpha, and bovine insulin did not compete with IL-1 beta. By covalent ligand cross-linking and electrophoretic analysis, only type I IL-1R, a protein of 80 kd, was detected on chondrocytes. By immunocytochemical analysis, IL-1R was identified at the cell membrane level, in both normal and OA chondrocytes. The presence of nuclear staining was also observed, but only in OA chondrocytes. Recombinant human IL-1 (alpha and beta) induced the secretion of stromelysin and collagenase in a dose-dependent manner. The IL-1 concentration required for half-maximal metalloprotease stimulation was 3-4 times lower in OA chondrocytes than in normal cells. CONCLUSION: These results indicate that OA chondrocytes have a higher sensitivity to the stimulation of metalloprotease synthesis by IL-1 than do normal cells. This could be related to the increased levels of IL-1R expressed in the OA cells. The implications of these findings with regard to the possible roles of IL-1 and IL-1R in the pathogenesis of OA are discussed.

Aged↗
PubMed1985

Fc epsilon receptors on rat B-lymphocytes: specificity and binding kinetics.

The binding kinetics of radiolabelled rat IgE to Fc epsilon receptors (R) of rat B-cells have already been studied in IgE-stimulated animals. The receptors expressed after Nippostrongylus brasiliensis infection or 2 injections of 5 mg IgE/100 g body wt were class-specific: IgE binding was not hindered by rat IgM, IgD, IgA, IgG1, IgG2a, IgG2b and IgG2c in immunocompetitive-binding assays. The rat B-cell Fc epsilon R were not species-specific, since mouse IgE competes with rat IgE for binding to these receptors. The apparent number of Fc epsilon R on rat mesenteric lymph node B-cells varied with temp and was 1.1-2.4 X 10(5) at 4 degrees C and 5.9-7.7 X 10(5) at 37 degrees C. The experimental Ka was not influenced by temp and had an average value of 1.38 X 10(8) M-1. At 4 degrees C the IgE binding to B-cell Fc epsilon R had an association rate of 4.9 X 10(3) M-1 sec-1 and a dissociation rate of 4.65 X 10(-5) sec-1. After a very strong stimulation produced by injecting 5 mg IgE/100 g body wt every 24 hr for 5 days, the equilibrium binding curve became diphasic, indicating a probable heterogeneity of the B-cell Fc epsilon R.

Animals↗
PubMed1987

Age-dependence and free fatty acid modulation of binding kinetics at the benzodiazepine binding site of serum albumin in neonates and adults determined using fast reaction methods.

Binding kinetics at the benzodiazepine binding site of albumin have been determined as a function of age and fatty acid concentration in serum from 32 neonates (umbilical cord blood), 28 adults aged 23-65 years and 24 adults aged 66-101 years using fluorescence-time curves obtained in a Durrum-Gibson stopped-flow apparatus and the specific marker ligand dansylsarcosine. The binding reaction can be described by a 2-step mechanism characterised by four constants: the association rate constant k2 and dissociation rate constant k-2 for the rate limiting step-2; the affinity constant KA' for the formation of an unstable intermediate complex in step-1; the affinity constant KA for the overall reaction. There were marked differences in k2 between the three groups of subjects with highest values occurring in neonates (mean: 296 s-1), intermediate values in adults aged 23-65 years (mean: 200 s-1, P less than 0.001) and lowest values in adults aged 66-101 years (mean: 144 s-1, P less than 0.001). The dissociation rate constants (k-2) averaged 18.5 s-1 overall, showed only minor changes with increasing age and corresponded to dissociation half-lives of 42 ms, 38 ms and 33 ms for neonates, adults 23-65 years and adults 66-101 years respectively. KA' in neonates and in the group of elderly subjects were lower than in adults aged 23-65 years.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Serpin-serine protease binding kinetics: alpha 2-antiplasmin as a model inhibitor.

We have examined in detail the kinetics of binding of the serpin alpha 2-antiplasmin to the serine proteases alpha-chymotrypsin and plasmin. These represent model systems for serpin binding. We find, in contrast to earlier published results with alpha 2-antiplasmin and plasmin, that binding is reversible, and slow binding kinetics can be observed, under appropriate conditions. Binding follows a two-step process with both enzymes, with the formation of an initial loose complex which then proceeds to a tightly bound complex. In the absence of lysine and analogues, equilibrium between alpha 2-antiplasmin and plasmin is achieved rapidly, with an overall inhibition constant (Ki') of 0.3 pM. In the presence of tranexamic acid or 6-aminohexanoic acid, lysine analogues that mimic the effects of fibrin, plasmin binding kinetics are changed such that equilibrium is reached slowly following a lag phase after mixing of enzyme and inhibitor. The Ki' is also affected, rising to 2 pM in the presence of 6-aminohexanoic acid concentrations above 15 mM. Thus extrapolation to the in vivo situation indicates that complex formation in the presence of fibrin will be delayed, allowing a burst of enzyme activity following plasmin generation, but a tight, pseudoirreversible complex will result eventually. Chymotrypsin is more weakly inhibited by alpha 2-antiplasmin, exhibiting an overall Ki' of 0.1 nM, after two-stage complex formation. The inhibition constant for the initial loose complex (Ki) is very similar for both enzymes. The difference in binding strength between the two enzymes is accounted for by the dissociation rate constant of the second step of complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminocaproic Acid↗
PubMed1990

Altered erythrocyte CR1 binding kinetics compensate for decreased binding capacity in rheumatoid arthritis.

Patients with rheumatoid arthritis have decreased numbers of CR1 per erythrocyte and decreased binding of immune complexes to erythrocytes. Overall erythrocyte immune complex binding activity depends on both the number and the binding kinetics of CR1. We measured kinetic parameters for the interaction between a complement-containing dsDNA:anti-dsDNA probe and erythrocytes in patients with rheumatoid arthritis and normal controls. The results indicate that: 1) the maximum quantity of immune complexes bound per erythrocyte was significantly decreased in rheumatoid arthritis compared with normal controls (p less than or equal to 0.009); 2) the steady state binding constant, Kss, and the association rate constant for binding of immune complexes to erythrocytes, ka, were significantly increased in rheumatoid arthritis versus normal controls (p less than or equal to 0.0001 and 0.002 respectively); 3) the dissociation rate constant for the release of bound immune complexes from erythrocytes, kd, was slightly smaller in rheumatoid arthritis but this difference was not statistically significant; and 4) the energies of activation for the association and dissociation reactions, Eaa, and Ead, did not differ between the two groups. These data confirm that while the maximum quantity of immune complexes bound per erythrocyte is decreased in rheumatoid arthritis, the association rate constants are larger and dissociation rate constants slightly smaller than those of normal controls. Changes in these kinetic parameters compensate for the decrease in the maximum quantity of immune complexes bound per erythrocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-Antibody Complex↗
PubMed1987

Evaluation of T4 and T3 binding kinetics in the thyroxine binding globulin abnormality of Australian aborigines.

Low serum total T4 associated with subnormal concentrations of thyroxine binding globulin (TBG) has been reported in up to 40% of euthyroid Australian aborigines. It has been suggested that these subjects show both diminished concentration of TBG and reduced TBG affinity for T4 (Sarne et al., 1985). We have compared 12 euthyroid aborigines with low T4 (total T4 44 +/- 5 nmol/l) and aborigines with normal T4 (T4 99 +/- 9 nmol/l, n = 12) using measurements of free T4 and T3 by equilibrium dialysis. TBG was measured both by RIA (Henning, Berlin, FRG) and a method dependent on T4 binding (Corning Immophase). Aborigines with low T4 showed lower levels of free T4 (12.6 +/- 0.6 cf. 18.7 +/- 1.0 pM), free T4 index (66 +/- 8 cf. 98 +/- 13), total T3 (1.1 +/- 0.2 cf. 1.6 +/- 0.3 nmol/l), TBG RIA (14.0 +/- 0.6 cf. 25.0 +/- 1.2 ng/l), and TBG Immophase (9.0 +/- 0.5 cf. 22.0 +/- 1.2 mg/l) (P less than 0.01), but free T3 (5.3 +/- 0.4 cf. 4.7 +/- 0.4 pM) and TSH (1.9 +/- 0.2 cf. 1.8 +/- 0.2 mU/l) were not significantly different from the values found in aborigines with normal T4. Scatchard analysis of T4 and T3 binding was performed using serum diluted 1 : 20,000 for T4 and 1 : 500 for T3 (barbitone buffer pH 8.6, 4 degrees C, dextran-coated charcoal separation). In euthyroid low T4 aborigines compared to those with normal T4, both T4 capacity (106 +/- 14 cf. 238 +/- 13 nM, P less than 0.01) and affinity (5.05 X 10(10) cf. 8.47 X 10(10) M-1, P less than 0.05) were significantly reduced. Similarly, both T3 capacity (62 +/- 10 cf. 154 +/- 16 nM, P less than 0.01) and affinity (1.67 X 10(9) cf. 2.28 X 10(9) M-1, P less than 0.02) were reduced. A substantial minority of euthyroid Australian aborigines have a TBG variant characterized by both reduced capacity and affinity of T4 and T3. These findings suggest that TBG may be both qualitatively and quantitatively abnormal in these subjects.

Alpha-Globulins↗
PubMed1985

Rapid method for quantitation of androgen binding protein in Sertoli cell cultures and its use for measurement of binding kinetics.

The accurate measurement of the kinetics of binding of 5 alpha-dihydrotestosterone to the Sertoli cell specific protein, androgen binding protein (ABP), has been frustrated by the extremely rapid rate of dissociation of the ABP-dihydrotestosterone complex. We describe a rapid and highly sensitive assay suitable for ABP quantitation which utilizes DEAE Bio-Gel and [3H]dihydrotestosterone. The assay has been used to accurately measure the rate of dissociation (8.25 X 10(-4) s-1, t1/2 14 min) and the rate of association (2.04 X 10(5) M s-1) of the binding of [3H]dihydrotestosterone to rat ABP. The ratio of these rate constants is in perfect agreement with the equilibrium dissociation constant determined by Scatchard analysis (4.0 nM). This multipoint assay is extremely rapid such that binding can be measured at equilibrium, it has high precision (coefficient of variation 3%), and is particularly useful at low protein concentrations (50 ng/ml); furthermore, the assay background of nonspecific 3H-binding is extremely low (0.2%). Since at such low protein concentrations a 10 point Scatchard analysis can be performed on 1 ml culture medium containing as little as 3 fmol ABP, the assay is suitable for monitoring changes in ABP secretion resulting from manipulations of cells in culture. The assay which utilizes DEAE Bio-Gel A is compared to five alternative methods: the standard method of steady state gel electrophoresis, Dextran-coated charcoal assay, hydroxylapatite assay, DEAE filter assay, and radioimmunoassay. The DEAE Bio-Gel assay has advantages over all of these alternative methods. In summary, this new assay is particularly useful for monitoring temporal changes in the secretion of ABP, and the method is equally effective in quantitating ABP in rat, rabbit and hamster Sertoli cell cultures.

Adsorption↗
PubMed1986

Interleukin-2 monoclonal antibody affinity adsorption. The critical role of binding kinetics for optimal immunoadsorption.

With the ready availability of monoclonal antibodies reactive with an extensive spectrum of antigens, immunoaffinity adsorption has become more widely applicable for protein purification. However, given several monoclonal antibodies reactive with the same antigen, most investigators have found that only a few antibodies are useful for solid-phase immunoaffinity antigen purification. Accordingly, in order to determine the parameters of monoclonal antibody-antigen binding most important for effective immunoaffinity adsorption, equilibrium and kinetic binding experiments were performed using radiolabeled interleukin-2 (IL-2) as antigen and four different IL-2-reactive monoclonal antibodies. The antibodies were found to differ primarily with respect to their kinetic binding characteristics; at 37 degrees C IL-2 bound to two of these antibodies very rapidly, while it bound to the other two more slowly. When binding was performed at 4 degrees C, the equilibrium dissociation constants for all of the antibodies decreased due to a more marked prolongation of the dissociation rate than the association rate. However, at 4 degrees C the association rates of the two slow-reactive antibodies became retarded so markedly that efficient affinity adsorption did not occur. By comparison, for both of these antibodies, efficient removal of IL-2 could be obtained if adsorption was performed at 37 degrees C, provided the column flow rate was adjusted according to the IL-2 association rate. Kinetic considerations also dictated IL-2 adsorption to mixtures of two or more monoclonal antibodies: IL-2 immunoadsorption correlated with the association rates of the individual antibodies, rather than the equilibrium binding constants. These results indicate that the most important parameter for efficient affinity adsorption is the association rate constant. In addition, the results obtained indicate that monoclonal antibodies may differ markedly as regards their kinetic binding characteristics, and that all antibodies can serve as effective immunoadsorbents, provided their antigen binding characteristics are known.

Antibodies, Monoclonal↗
PubMed1992

Drug-protein binding kinetics in patients with type I diabetes.

Sera from 17 patients with Type I diabetes and 19 healthy volunteers have been examined to evaluate whether the kinetics of the binding of drugs to Site II of serum albumin is altered in diabetes. Stopped-flow measurements showed that the association velocity and the affinity constants of the fluorescent marker dansylsarcosine were significantly lower in diabetes (160 s-1 and 2.0 x 10(5) l.mol-1) than in non-diabetics (196 s-1 and 4.0 x 10(5) l.mol-1). The dissociation velocity was not different [20.3 s-1 vs. 19.4 s-1]. Although patients with a reduced albumin concentration were excluded the diabetics had significantly lower concentrations than the healthy volunteers. There was a significant correlation between decreased glycosylation of albumin and increased association velocity. The dissociation velocity constants were correlated with the molar concentration ratio of free fatty acids/human serum albumin. Thus, the extent of glycosylation and the amount of fatty acids bound per mole albumin can both affect the kinetics of drug binding to Site II. The lower affinity in patients with Type I diabetes is due to the increased in the glycoalbumin concentration.

Adult↗
PubMed1987

Effect of flutamide on hepatic cytosolic methyltrienolone (R1881) binding kinetics and testosterone responsive hepatic drug and steroid metabolism in the adult male rat.

Flutamide was used to investigate the mechanism involved in androgen responsive hepatic microsomal drug and steroid metabolism. We compared the antiandrogenic action of flutamide on the prostate to its effect on testosterone responsive hepatic microsomal benzo[a]pyrene hydroxylase (BPH) and testosterone reductase (TR) activities. Male Wistar rats, castrated as adults, were treated with 5 mumoles.kg-1.day-1 of testosterone enanthate subcutaneously for 10 days. Co-administration of increasing doses of flutamide caused a dose-dependent reduction in prostate to body weight ratios and, in the same animals, caused significant alterations in adult male hepatic microsomal BPH and TR activities. These doses of flutamide did not affect the serum testosterone levels. To test the possibility that the action of flutamide on androgen responsive hepatic microsomal drug and steroid metabolism may be similar to that occurring in the prostate, a tissue which contains an androgen receptor, we also studied the effect of flutamide on the binding kinetics of the high affinity hepatic cytosolic [3H]R1881 binding protein in vivo. Scatchard analysis of [3H]R1881 binding data revealed a reduction in the binding capacity of the hepatic cytosolic androgen binding protein in castrated animals treated with a combination of flutamide and testosterone enanthate at doses capable of maximally altering hepatic microsomal drug and steroid metabolism. No alteration in binding affinity occurred in this treatment group. However, a decreased binding affinity was found when flutamide alone was given. The binding kinetics of the hepatic cytosolic androgen binding protein were not altered in the castrated adult male with or without testosterone treatment. When flutamide was injected daily into the intact adult female rat, no effect was observed on either hepatic microsomal BPH or TR activities. Taken together, these data indicate that flutamide reduces hepatic cytosolic R1881 binding in the adult male rat, and this may explain some of the effects of this antiandrogen on testosterone-sensitive hepatic microsomal drug and steroid metabolism.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1992

Effects of diltiazem and verapamil on (+)-PN 200-110 binding kinetics in dog cardiac membranes.

The effects of d-cis-diltiazem (diltiazem) and verapamil on 1,4-dihydropyridine binding to dog cardiac membranes were studied in competition, saturation and kinetic binding experiments with [3H](+)-PN200-110. Diltiazem increased [3H](+)-PN200-110 binding with an observed maximal effect at 50 microM, while verapamil decreased [3H](+)-PN200-110 binding in a dose-dependent manner. Scatchard analysis of saturation binding data revealed that diltiazem (50 microM) increased the maximal binding site density and verapamil (100 microM) increased the dissociation constant (KD) of [3H](+)-PN200-110 binding. The kinetic experiments demonstrated that diltiazem significantly reduced both the association and the dissociation rate of [3H](+)-PN200-110 binding, resulting in no significant change in the apparent KD. In contrast, verapamil accelerated dissociation and slowed down association of [3H](+)-PN200-110 binding. Diltiazem appears to alter both the number of [3H](+)-PN200-110 binding sites and the characteristics of [3H](+)-PN200-110 binding.

Allosteric Regulation↗
PubMed1986

A comparison of microsomal and synaptic delta site binding kinetics.

High-affinity binding of the agonist, [3H]D-ala2-D-leu5-enkephalin (DADLE), to delta sites on bovine hippocampal synaptic plasma membranes (SPM) entails a multi-step association process. Microsomal binding sites, which are thought to originate from internal membranes (golgi, ser, etc.), display kinetic patterns that differ from SPMs. This is evidenced by the absence of an association time dependent rate of dissociation from microsomal binding sites. Although high affinity steady state binding of agonists to microsomes occurs, kinetic analysis indicates little or no formation of the high affinity slowly dissociating complex. This slowly dissociating complex of SPMs is most sensitive to guanine nucleotides. Consequently, the effect of Gpp(NH)p on dissociation is significantly less for microsomes.

Animals↗
PubMed1991

Distinct colchicine binding kinetics of bovine brain tubulin lacking the type III isotype of beta-tubulin.

In mammalian brain, beta-tubulin occurs as a mixture of four isotypes designated as types I, II, III, and IV. It has been speculated in recent years that the different tubulin isotypes may confer functional diversity to microtubules. In an effort to investigate whether different tubulin isotypes differ in their functional properties we have studied the colchicine binding kinetics of bovine brain tubulin upon removal of the beta III isotype. We found that the removal of the beta III isotype alters the binding kinetics from biphasic to monophasic with the disappearance of the slow phase. The kinetics become biphasic with the reappearance of the slow phase when the beta III-depleted tubulin was mixed with the beta III fraction eluted from the affinity column with 0.5 M NaCl. The analysis of the kinetic data reveals that the tubulin dimers containing beta III bind colchicine at an on-rate constant of 35 M-1 s-1 while those lacking beta III bind at 182 M-1 s-1. Our results strongly suggest that the beta-subunit plays a very important role in the interaction of tubulin with colchicine.

Animals↗
PubMed1988

Binding kinetics of delta opioid receptors differ for microsomal and synaptic sites.

Earlier, we demonstrated that agonist binding to synaptic plasma membranes involves a multi-step association process. In this study, high affinity binding kinetics of an agonist, [3H]D-Ala2-D-Leu5-enkephalin (DADLE), to delta sites on bovine hippocampal microsomal and synaptic plasma membranes (SPM) were compared. delta site selectivity of DADLE was ensured by suppressing undesirable mu site binding with 20 nM unlabeled D-Ala2-MePhe4-Glyol5-enkephalin. The kinetics of receptor binding to microsomal delta sites are generally more rapid than those of SPMs. Furthermore, the association time-dependent rate of dissociation, which is readily observed with SPMs, was not detected for microsomal binding sites. Although the apparent KD of DADLE did not differ significantly from that in SPMs, kinetic analysis indicated that little or no formation of the high affinity, slowly dissociating, complex occurred with microsomes. The absence of this complex, shown previously in SPMs to be most sensitive to guanine nucleotides, appeared to account for the attenuated effect of guanyl 5'-yl-imidodiphosphate [Gpp(NH)p] on dissociation from microsomes. Nevertheless, the presence in microsomes of inhibitory guanine nucleotide binding proteins was demonstrated by specific 32P-labeling by pertussis toxin of bands at 39 and 41 kDa, attributable to the alpha subunit of Go and Gi, respectively. The action of 100 mM Na+ to increase the off-rate is similar for both preparations. In contrast, addition of Mn2+ reduced the rates of association and dissociation for both subcellular fractions. The off-rate in the presence of Mn2+ is similar for SPMs and microsomes, displaying association time-dependent rates of dissociation for both. To determine whether Mn2+ promotes coupling in microsomes, the effect of Gpp(NH)p was examined. After a 60-min association, Gpp(NH)p did not affect microsomal kinetics but increased the off-rate from SPMs. The actions of both Na+ and Mn2+ appear to be mediated at early steps in the association process.

Animals↗
PubMed1985

Urinary protein binding, kinetics, and dynamics of furosemide in nephrotic patients.

The urinary protein binding, kinetics, and dynamics of furosemide were studied in five nephrotic patients after intravenous dosing. Serial plasma and urine samples containing furosemide were analyzed by HPLC, and drug binding to plasma and urinary proteins was determined using equilibrium dialysis techniques. In comparison to data reported previously in healthy subjects, the steady-state volumes of distribution and nonrenal plasma clearances were significantly increased in nephrotic patients, reflecting the reduced binding of furosemide to plasma proteins. Although there was no significant difference in renal clearance between these two groups, the unbound renal clearance of furosemide was significantly reduced in nephrotic patients even when compensated for by the number of functioning nephrons. Furosemide was extensively bound to urinary protein (19.6-78.4%), and the binding was dependent on the degree of proteinuria. Nevertheless, dose-response analyses, in which the response was represented by sodium excretion rate and the dose by urinary excretion rate of unbound drug, demonstrated that nephrotic patients were less responsive to equivalent amounts of unbound diuretic as compared to healthy subjects.

Adult↗
PubMed1989

Dopamine D1 receptors labelled with [3H]SCH23390 in rabbit cerebral cortex and neostriatum. Equilibrium binding, kinetics and selectivity.

The binding characteristics of the novel benzazepine compound SCH23390 were studied using membrane preparations from rabbit cerebral cortex (CTX) and neostriatum (CPU; caudate putamen). The association kinetics of [3H]SCH23390 to membranes from CTX and CPU were rapid, while the dissociation kinetics were extremely slow and only around 40-60% of the binding was displaced two hours after the addition of either S(+)-butaclamol or 30 volumes of buffer. The saturation curves revealed that [3H]SCH23390 bound with high affinity in both tissues, with densities of 133 fmol/mg protein for CTX (Kd 25 degrees C = 0.31 nM) and 664 fmol/mg protein for CPU (Kd = 0.13 nM). the specificity of binding to the cortical D1 receptor was verified in competition experiments with a variety of dopaminergic agents. The rank order of potency of these compounds was consistent with the pharmacology of the dopaminergic D1 site. All competition curves were better fitted to a one-site model with Hill coefficients around one, indicating that [3H]SCH23390 was binding to a single cortical site. The stereoselectivity of the cortical [3H]SCH23390 binding site could be demonstrated by the use of enantiomer pairs of dopaminergic drugs. This study provides compelling evidence that [3H]SCH23390 binds to dopamine D1 receptors in the neostriatum and cerebral cortex of the rabbit.

Animals↗
PubMed1987

Stabilization mechanism of prostacyclin by human serum: an approach by binding kinetics using a stable prostaglandin I2 analogue, iloprost.

We used a gel filtration method and a stable prostaglandin I2 (prostacyclin) analogue, iloprost, to study the kinetics of prostaglandin I2 binding by human serum proteins. Binding equilibrium experiments conducted at physiological prostaglandin I2 concentration (nM) yielded a KD of 10(-9) and a capacity of approx. 50 nM for the serum binding protein(s). Kinetic measurements gave a dissociation rate constant of 10(-3) s-1. When binding equilibrium was established at various ligand concentrations ranging from nM to microM, a result indicating an unsaturable binding was obtained utilizing this method. On the other hand, saturation was achieved with a ligand concentration as high as 50-100 microM by another binding method. A KD of 7 X 10(-5) and a capacity of approx. 600 microM was obtained. This apparent discrepancy was resolved by performing parallel experiments using purified human serum albumin samples and serum. It is concluded that the large quantity of serum albumin, approx. 600 microM, in serum may compensate for its low KD (approx. 10(-5] for prostaglandin I2, thus simulating a binding protein with a KD of 10(-9) and a limited capacity. These data offer direct information regarding how prostaglandin I2 is stabilized by serum and is transported to the platelet prostaglandin I2 receptors. There is a strong implication that serum albumin is the major if not the only protein responsible for binding of prostaglandin I2.

Blood Proteins↗